DESCRIPTION (provided by applicant: Diarrheal diseases kill 2.2 million people annually, mostly children, and are the second largest cause of death worldwide. Vibrio cholerae, Salmonella, and the enteropathogenic and enterotoxigenic E. coli (EPEC and ETEC respectively) cause diarrheal diseases that are devastating to people in developing countries and also pose a serious threat to international travelers and military personnel. In addition these organisms present a health threat here in the United States and are listed as Category B agents of bioterrorism by the Centers for Disease Control. The virulence of these organisms is attributed, in part, to the Type IV pili, which allow the bacteria to form microcolonies and colonize the human intestine. Our x-ray crystallographic results on the pilin subunits that comprise the pili suggest that the two Type IV pilin subclasses, IVa and IVb, are structurally distinct. We generated a molecular model for the Type IVb toxin coregulated pilus (TCP) from V. cholerae by integrating crystal structure and packing data with the filament dimensions and helical symmetry of the TCP filaments. This model is generalizable for all Type IV pili whereby the conserved N-terminal a- helices anchor the structurally variable pilin heads, which contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion and microcolony formation. In this application we intend to structurally characterize Type IVb pili from the enteric pathogens V. cholerae, S. enterica serovar Typhi, EPEC and ETEC. We plan to solve the crystal structures of several N-terminally truncated Type IVb pilins, and to determine their orientation in the pilus filaments using a newly emerging and powerful method of structural analysis, deuterium exchange mass spectrometry. These data will be integrated to derive molecular models for the Type IVb pili using the TCP model as a template. Knowledge of the structure and assembly of these key virulence factors will provide insight into their colonization functions and a basis for the design of vaccines and therapeutics. [unreadable] [unreadable]